Pharmaceutical composition

ABSTRACT

Polypeptide pharmaceutical compositions having improved stability and resistance to aggregation, particle formation and precipitation comprising a polypeptide pharmaceutical and poloxamer surfactants alone, or in combination with polysorbate surfactants. Preferred polypeptides stabilized are bactericidal/permeability increasing (BPI) protein, biologically active fragments of BPI, biologically active analogs of BPI, and biologically active variants of BPI.

This is a continuation of U.S. application Ser. No. 08/472,995, filedJun. 7, 1 995, now U.S. Pat. No. 5,696,090, which is a Divisional ofU.S. application Ser. No. 08/190,869 filed Feb. 2, 1994, U.S. Pat. No.5,488,034, which is a continuation-in-part of U.S. patent applicationSer. No. 08/012,360 filed Feb. 2, 1993 abandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to pharmaceutical compositionsand more specifically to improved protein and polypeptidepharmaceuticals for use as parenteral drugs. Recent advances in thedevelopment of genetic engineering technology have made a wide varietyof biologically active polypeptides available in sufficiently largequantities for use as drugs. Polypeptides, however, can be subject toparticulate formation and loss of biological activity by a variety ofchemical and physical means including denaturation due to heating orfreezing and by exposure to extreme pH or other chemical degradation.

Particulate formation and loss of biological activity can also occur asa result of physical agitation and interactions of polypeptide moleculesin solution and at the liquid-air interfaces within storage vials. It isbelieved that the polypeptide molecules adsorb to an air-liquidinterface, unfolding to present hydrophobic groups to air with thehydrophilic groups immersed in the aqueous phase. Once so positioned atthe surface, the polypeptide molecules are susceptible to aggregation,particle formation and precipitation. It is also believed that furtherconformational changes can occur in polypeptides adsorbed to air-liquidand solid-liquid interfaces during compression-extension of theinterfaces such as occurs from agitation during transportation orotherwise. Such agitation can cause the protein to entangle, aggregate,form particles and ultimately precipitate with other adsorbed proteins.

Particle formation due to surface denaturation can be somewhatcontrolled by appropriate selection of the dimensions of storage vialsand by minimizing the air volume (headspace) in those vials. In thisregard, partially filled containers represent the worst case forvibration induced precipitation.

Particle formation can also be controlled by incorporation ofsurfactants into the protein containing composition in order to lowerthe surface tension at the solution-air interface. Classic stabilizationof pharmaceuticals by surfactants or emulsifiers has focused on theamphipathic nature of molecular groups containing both hydrophilic andhydrophobic properties within the surfactant molecule. Thus, the artteaches that one can make a stable solution of immiscible molecules suchas oil-in-water or water-in-oil by selecting an appropriate surfactantas a compatibilizer. One example is the stable emulsification of soybeanoil using poloxamer 188 (PLURONIC F-68, BASF Wyandotte Corp.,Parsippany, N.J.). Another example is the use of polysorbate 80 (TWEEN80, ICI Americas, Inc., Wilmington, Del.) to emulsify oil-solublevitamins A, E and K in aqueous solution for administration via oral andvascular routes. Work by Krantz, et al., "Sugar Alcohols--XXVIII.Toxicologic, Pharmacodynamic and Clinical Observations on TWEEN 80,"Bull of the School of Med., U. of Md., 36, 48 (1951) laid the groundworkleading to the listing of polysorbate 80 as a drug ingredient for whichUSP/NF requirements have been established in U.S. Pharmacopeia XXII.

Of interest to the present invention is the work related to use ofpolysorbate 80 for stabilization of antibody-based product formulationsas described in Levine, et al., J. Parenteral Sci. Technol., 45, 3,160-165 (1991). This work disclosed that the amount of surfactantrequired for stabilization was in excess of the theoretical minimumrequired to reduce surface tension. The work further showed that theneed for excess surfactant beyond the theoretical minimum could beattributed to (1) the concentration required to maintain an intactprotective layer on a turbulent interface during random shaking; and (2)to surfactant loosely associated with protein and bound to containerwalls.

Regulatory requirements limit the types and specific identities ofsurfactants that can be incorporated into parenteral compositions forinjection into the human body. Generally accepted surfactants having ahistory of use and listed in the U.S. Pharmacopoeia XXI includepoloxamer and polysorbate polymers. However, either of these alone mayprovide less than complete stabilization for the pharmaceuticalcompositions when used at concentrations of 0. 1% or lower. Elevatedconcentrations of surfactant may pose increased risk of toxic effects,earlier onset of hemolysis, and observed changes in neutrophils andplatelets, both of which are involved in blood complement activation.The highest safe concentration for poloxamer 188 in approved parenteralsolutions is 2.7% when it is used in limited doses as a blood substituteand is diluted as much as 10 fold in the bloodstream. Similarly,polysorbate 80, approved in parenteral solutions for over 20 years, israrely used in concentrations greater than 0. 1% in solution volumes of100 mL or more. Krantz et al., supra, identifies the onset of hemolysisin the dog for a polysorbate concentration of 0.1% at 90 minutes.Neonatal deaths have been associated with the use of polysorbate 80 atconcentrations of greater than 1%. Accordingly, there exists a need inthe art for pharmaceutical compositions providing improved proteinstability which comprise only those components which are regarded assafe and are included in parenterals approved by regulatory authoritiesfor commercial use.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions ofpolypeptides and is directed to the discovery that poloxamer surfactantsand combinations of poloxamer surfactants with polysorbate surfactantsenhance the solubility/stability of bactericidal/permeability increasing(BPI) protein, biologically active fragments of BPI, biologically activeanalogs of BPI, and biologically active variants of BPI (produced byeither recombinant or nonrecombinant means) in aqueous solution. Theinvention particularly provides for solubilization/stabilization ofbactericidal/permeability increasing proteins which are biologicallyactive amino-terminal fragments of BPI or analogs and variants thereof.Amino-terminal fragments of BPI, such as those designated rBPI₂₃ or anyamino-terminal fragment comprising from about the first 193 to about thefirst 199 amino-terminal amino acid residues of BPI, are believed to beparticularly susceptible to loss of stability in aqueous solution.

The present invention is directed in particular to the discovery that acombination of two specific types of surfactants provides a surprisingimprovement in protein stability to pharmaceutical compositions comparedto either surfactant alone. Specifically. it has been found that apharmaceutical composition comprising the combination of a poloxamer(polyoxypropylene-polyoxyethylene block copolymer) surfactant andpolysorbate (polyoxyethylene sorbitan fatty acid ester) surfactantprovides improved stability and resistance to aggregation, particleformation and precipitation of protein pharmaceutical agents. Thecombination of these two types of surfactants provides improvedstability and resistance to surface denaturation aggregation, particleformation and precipitation compared with either surfactant alone.

The poloxamer surfactant component is preferably present in aconcentration of from about 0.01% to about 1% by weight with aconcentration of 0.1% to 0.2% by weight being preferred to stabilizeprotein solutions comprising less than or equal to 2 mg/mL. Thepolysorbate surfactant component is preferably present in aconcentration of from about 0.0005% to about 1% by weight with aconcentration of 0.002% by weight being preferred. Most preferred is thecombination comprising 0.1% to 0.2% by weight of poloxamer 188 and0.002% by weight polysorbate 80. This combination is particularly usefulfor preventing particle formation of extremely degradation sensitiveproteins such as bactericidal/permeability increasing protein (BPI) butis also useful for promoting the stability of other polypeptidepharmnaceuticals. It is contemplated that the combination of poloxamerand polysorbate surfactants may be used alone or in combination withadditional surfactants. Moreover, the invention is not limited to asingle poloxamer surfactant in combination with a single polysorbatesurfactant and can include one or more poloxamer surfactants incombination with one or more polysorbate surfactants.

A further aspect of the invention relates to the discovery that apoloxamer surfactant is particularly useful for thesolubilization/stabilization of compositions comprising an aqueoussolution of BPI protein or biologically active fragments, analogs, orvariants of BPI protein (produced by recombinant or nonrecombinantmeans). The invention provides a method of solubilizing/stabilizing suchpolypeptides by contacting the polypeptide with a poloxamer surfactant.Without being bound by a theory of the invention, it is believed thatpoloxamer surfactants stabilize BPI protein products not by a mechanisminvolving lowering the surface tension of the aqueous solution, but, atelevated temperatures, by stabilizing unfolded and partially unfoldedBPI protein molecules and preventing precipitation of those molecules.

Preferred poloxamer surfactants are characterized by a HLB value greaterthan about 14 and a surface tension between 10 and 70 mN/m as measuredin aqueous solution at room temperature and at a concentration of 0.1%.More preferred is a poloxamer surfactant which has an HLB value betweenabout 25 and 35 and has a surface tension between 30 and 52 mN/m asmeasured in aqueous solution at room temperature and at a concentrationof 0.1%. Most preferred is poloxamer 188 available commercially asPLURONIC F-68 (BASF Wyandotte, Parsippany, N.J.) which is characterizedby a surface tension of 50 mN/m and by an HLB value of 29.

A preferred polysorbate surfactant preferably has a surface tensionbetween 10 and 70 mN/m as measured in aqueous solution at roomtemperature and at a concentration of 0. 1%. More preferably, thepolysorbate surfactant is characterized by a hydrophilic/lipophilicbalance (HLB) value of about 15 and by a surface tension between 40 and50 mN/m as measured in aqueous solution at room temperature and at aconcentration of 0. 1%. Most preferred is polysorbate 80 (sorbitanmono-9-octadeconoate) which is available commercially as TWEEN 80 (ICIAmericas Inc., Wilmington, Del.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting survival results over time of anactinomycin-D sensitized mouse model:

FIG. 2 is a graph depicting survival results according to BPI dose in anactinomycin-D mouse model; and

FIG. 3 is a graph depicting turbidity measurements of various BPIproteins with and without the preferred surfactants of the invention.

FIG. 4 is a graph depicting surface tension measurements of rBPI₂₁ Δcyssolutions with varying surfactant concentrations of polysorbate 80(PS80) and poloxamer 188 (F68).

FIG. 5 is a series of graphs of differential scanning calorimetryresults of rBPI₂₁ Δcys with various concentrations of the surfactantpoloxamer 188 (F68).

FIG. 6 is another series of graphs of differential scanning calorimetryresults of rBPI₂₁ Δcys with various concentrations of poloxamer 188(F68)

FIG. 7 is a plot of the denaturation and precipitation temperatures ofrBPI₂₁ Δcys over varying concentrations of the surfactant poloxamer 188(F68).

FIG. 8 is a series of graphs of differential scanning calorimetryresults of rBPI₂₁ Δcys with various concentrations of polysorbate 80(PS80) alone or in combination with 0.1% poluxamer 188 (F68) by weight.

FIG. 9 is a set of graphs of differential scanning calorimetry resultsof rBPI₂₁ Δcys with the surfactant polysorbate 80 (PS80) at twodifferent concentrations.

FIG. 10 is a set of graphs of differential scanning calorimetry resultsafter a solution of rBPI₂₁ Δcys and poloxamer 188 (F68) was heated to atemperature higher than the denaturation/unfolding temperature but lowerthan the precipitation temperature, and then was cooled down for repeatscanning.

DETAILED DESCRIPTION

The present invention provides improved methods and materials formaintaining the stability of polypeptide pharmaceuticals and preventingsurface denaturation of such biologically active polypeptides.Specifically, the invention relates to the discovery that a combinationof two specific types of surfactant molecules provides synergisticimprovements in stabilization from surface denaturation of polypeptidepharmaceuticals. The invention also relates to the discovery thatpoloxamer surfactants have unique properties in thesolubilization/stabilization of BPI-related proteins. While specificembodiments of the invention are directed to stabilization ofbactericidal/permeability increasing protein (BPI) and biologicallyactive fragments and/or analogs or variants thereof which areparticularly susceptible to denaturation and particle formation, theutility of the invention extends generally to all protein andpolypeptide pharmaceuticals. BPI and active fragments and analogsthereof useful with the present invention include recombinant producedproteins such as described in U.S. Pat. No. 5,198,541. Co-owned,copending patent application Theofan et al., U.S. Ser. No. 08/064,693filed May 19, 1993, now U.S. Pat. No. 5,643,570, which is acontinuation-in-part application of U.S. Ser. No. 07/885,911 filed May19, 1992, abandoned, addresses BPI-Immunoglobulin fusion proteins whichare variants of BPI protein comprising at the amino terminal a BPIprotein or a biologically active fragment thereof, and retaining thesame biological activity of BPI protein. Particularly preferred BPImaterials include recombinant produced polypeptides produced accordingto the method of co-owned and copending Theofan et al. U.S. applicationSer. No. 08/013,801 filed Feb. 2, 1993, now U.S. Pat. No. 5,420,019, andentitled "Stable Bactericidal/Permeability-Increasing Protein Productsand Pharmaceutical Compositions Containing the Same," the disclosure ofwhich is herein incorporated by reference. A preferred BPI fragment ischaracterized by about 1 to 199 or about 1 to 193 of the amino-terminalamino acid residues of the mature human BPI molecule as set out in Grayet al., J. Biol. Chem., 264, 9505-9509 (1989) except that residue 185 isglutamic acid rather than lysine as specified in Gray. The recombinantexpression product of DNA encoding BPI amino acids 1 to 199 has beendesignated rBPI₂₃. The recombinant expression product of DNA encodingBPI amino acids 1 to 193 has been designated rBPI(1-193). A preferredBPI fragment analog comprises the first 193 amino acid residues as setout in Gray except that residue 185 is glutamic acid rather than lysineand the cysteine at position 132 is replaced with a non-cysteine residuesuch as alanine. Such a protein is designated rBPI₂₁ Δcys orrBPI(1-193)ala¹³².

EXAMPLE 1

In this example, tests of various surfactant systems were conducted todetermine their utility for surface stabilization of a polypeptidepharmaceutical (rBPI₂₃). The rBPI₂₃ was provided at a concentration of 1mg/mL in citrate buffered saline (0.02M citrate, 0.15M NaCl, pH 5.0).Various surfactants were then added to this preparation in order todetermine their utility as stabilizers.

According to this test, rBPI₂₃ BR-1! characterized by about 1 to about199 of the first 199 amino acids of the mature human BPI molecule andproduced according to the methods of Theofan et al., U.S. patentapplication Ser. No. 08/013,801 filed Feb. 2, 1993 was filled by hand to5 mL in sealed sterile 6 mL molded glass vials (total capacity 8.4 mL,Wheaton) in the desired formulation buffer. The vials to be tested wereset horizontally on a flat bed shaker (S/P rotor V) and fixed to theshaker by tape. Vials were then shaken at 150 rpm at room temperature.At 0 hours, 2-4 hours, and 18 hours, 150 μl samples were withdrawn in abiosafety cabinet using a 1 mL syringe fitted with a 21 gauge needle.The starting, in process, and ending soluble rBPI₂₃ concentrations weredetermined by an ion exchange HPLC assay and visual observation ofcloudiness of the solution was also recorded. The results are shownbelow in Table 1 in which acceptable stability was determined by visualinspection after the shake test.

Testing of protein preparations comprising single surfactants showedgood results for use of octoxynol-9 (TRITON X-100, Rohm & Haas),laureth-4, (BRIJ 30, ICI Americas), poloxamer 403 (PLURONIC P123, BASFWyandotte) and telomere B monoether with polyethylene glycol (ZONYLFSO-100, E.I. DuPont de Nemours). While these surfactants are capable ofreducing surface tensions to low levels, they are not included inapproved parenteral pharmaceuticals due to suspected toxic effects orunknown biocompatibility.

Testing of other surfactants as shown in Table 1 shows that surfactantsproducing a surface tension lower than 35 mN/m are capable ofstabilizing rBPI at surfactant concentrations of 0. 1%. This examplefurther shows that both polysorbate 80 (TWEEN 80) and poloxamer 188(PLURONMC F-68) were incapable of stabilizing the protein preparationalone under the shake test conditions employed. The incorporation ofpolysorbate 80 did, however, have the effect of clarifying a cloudysolution of BRIJ 30 which is not readily water soluble without the helpof an additional solubilizer.

                                      TABLE 1    __________________________________________________________________________            Surface Tension            mN/m at            0.1% Conc. at                    Surfactant     rBPI.sub.23 Conc.                                             Stability as            Room Temp. in                    Concentration                           Visual  by HPLC   Determined by    Exp       Surfactant            Water (w),                    in Form.                           Observation                                   (mg/mL)   Visual    No.       Used Buffer (b).sup.1                    Buffer                          3-4 hr                               18 hr                                   0 hr                                      3-4 hr                                          18 hr                                             Inspection    __________________________________________________________________________    1  ZONYL            17.sup.(w)                    0.100% --  Clear                                   0.96                                      --  1.00                                             Stable       FSO-100    2  PS-80            41.sup.(b)                    0.100% --  Cloudy                                   1.11                                      --  0.02                                             Unstable    3  BRIJ 30            27.5.sup.(b)                    0.500% Cloudy                               Cloudy                                   1.08                                      --  1.14                                             BRIJ 30 alone                                             is cloudy.    4  TRITON            32.sup.(b)                    0.100% Clear                               Clear                                   1.00                                      1.01                                          0.98                                             Stable       x-100    5  PLUR 34.3.sup.(w)                    0.100% Clear                               Clear                                   1.08                                      1.08                                          1.08                                             Stable       P123    6  BRIJ --      0.1%/  Clear                               Clear                                   1.19                                      1.21                                          1.17                                             Stable       30/PS-80     0.125%    7  PLUR 46.sup.(b)                    0.100% Clear                               Haze                                   1.23                                      1.22                                          0.95                                             Marginal       F-68                                  stability.                                             Slight haze,                                             specks.    8  PLUR 44.sup.(b)                    0.200% Clear                               Haze                                   -- --  1.04                                             Marginal       F-68                                  Stability.                                             SIight haze                                             with a few                                             specks.    9  PLUR 47.sup.(b)                    0.1%/  Clear                               Clear                                   1.14   1.09                                             Stable.       F-68/        0.001%                   Crystal clear       PS-80                                 with a few                                             specks.    __________________________________________________________________________     Surface tensions with superscript w are obtained from the surfactant     manufacturer. Surface tensions with superscript b are obtained     experimentally using Wilhelny plate method.

EXAMPLE 2

In this example, additional comparisons were carried out according tothe methods of Example 1 using various surfactants alone and incombination to stabilize a rBPI₂₃ preparation. The results are shownbelow in Table 2 in which acceptable stability was determined by visualinspection after the shake test. The results, particularly those ofexperiments 52-58 show the unexpected utility of the combination ofpoloxamer 188 and polysorbate 80 for stabilizing the rBPI₂₃ compositionat concentrations where either surfactant alone is incapable ofequivalently stabilizing the material under the conditions of the test.The experiments show that various combinations of concentrations of thetwo surfactants exhibit synergistic effects but that the preferredcombination specific to rBPI₂₃ at 1 mg/mL concentration is that having0.1% by weight poloxamer 188 and 0.001% by weight polysorbate 80 incitrate buffered saline (0.02M citrate, 0.15 NaCl, pH 5.0). The resultswith polysorbate 80 at concentrations lower than 0.001% produced promptcloudiness after 18 hours of shaking, but with only a small loss ofprotein as determined by ion-exchange HPLC MA7C column (Bio-Rad,Hercules, Calif.). Nevertheless, the cloudiness is unacceptable forappearance and suggests lowered stability. Testing with polysorbate 80at concentrations of 0.005% and above all give good stability at up to18 hours of shaking with little sign of protein loss by HPLC.Nevertheless, these higher concentrations of polysorbate 80 may provideless stability during long-term storage at 4° C. and at stresstemperatures of ambient room temperature or above.

                                      TABLE 2    __________________________________________________________________________                                Conc.     Stability as               Surfactant                      Visual    by HPLC   Determined    Exp        Conc. in Form.                      Observation                                (mg/mL)   by Visual    No.       Surfactant Used               Buffer 3-4 hr                           18 hr                                0 hr                                   3-4 hr                                       18 hr                                          Inspection    __________________________________________________________________________    1  ZONYL   0.100% --   Clear                                0.96                                   --  1.00                                          Stable       FSO-100    2  PS-80   0.100% --   Cloudy                                1.11                                   --  0.02                                          Unstable    3  Dextran Sulfate               1 mg/mL                      --   Cloudy                                -- --  0.00                                          Unstable    4  Glycerol               10.0%  --   Cloudy                                0.86                                   --  0.02                                          Unstable    5  HSA     5.0%   --   Cloudy                                0.92                                   --  0.00                                          Unstable    6  Control-               --     --   Cloudy                                1.13                                   --  0.03                                          Unstable       5 mL Fill       Volume    7  Control --     --   Clear                                1.13                                   --  1.04                                          Stable. One       8.4 mL                             speck of pre-       (complete)                         cipitate.       Fill Volume    8  Control-               --     Cloudy                           Cloudy                                1.16                                   0.21                                       0.00                                          Unstable       5 mL (partial)       Fill Volume    9  TRITON  0.500% Clear                           Clear                                1.04                                   0.99                                       1.11                                          Stable       X-100    10 PS-80   0.500% Clear                           Cloudy                                1.12                                   0.95                                       0.59                                          Unstable    11 PLURONIC               0.500% Clear                           Clear                                1.15                                   --  1.13                                          Stable       P123    12 BRIJ 30 0.500% Cloudy                           Cloudy                                1.08                                   --  1.14                                          BRIJ 30 alone                                          is cloudy.    13 TRITON  0.100% Clear                           Clear                                1.00                                   1.01                                       0.98                                          Stable       X-100    14 TRITON  0.010% Slt. Haze                           Cloudy                                0.96                                   0.84                                       0.04                                          Unstable       X-100    15 PLURONIC               0.100% Clear                           Clear                                1.08                                   1.08                                       1.08                                          Stable       P123    16 PLURONIC               0.100% Clear                           Clear                                1.23                                   1.26                                       0.94                                          Stable       P123    17 PLURONIC               0.050% Clear                           Slt. Haze                                1.21                                   1.18                                       1.11                                          Unstable       P123    18 PLURONIC               0.010% Cloudy                           Cloudy                                1.14                                   0.00                                       0.00                                          Unstable       P123    19 BRIJ 30/               0.1%/  Clear                           Clear                                1.19                                   1.21                                       1.17                                          Stable       PS-80   0.125%    20 BRIJ 30/               0.075%/                      Clear                           Clear                                1.22                                   1.20                                       1.18                                          Stable       PS-80   0.094%    21 BRIJ 30/               0.03%/ Slt. Haze                           Cloudy                                1.20                                   1.05                                       0.41                                          Unstable       PS-80   0.038%    22 BRIJ 30/               0.01%/ Cloudy                           Cloudy                                1.14                                   0.48                                       0.00                                          Unstable       PS-80   0.013%    23 PLURONIC               0.100% Clear                           Slt. Haze                                1.23                                   1.22                                       0.95                                          Marginal       F68                                Stability    24 PLURONIC               0.100% Clear                           Slt. Haze                                -- --  1.00                                          Marginal       F68                                Stability    25 PLURONIC               0.150% Clear                           Slt. Haze                                -- --  1.06                                          Marginal       F68                                Stability    26 PLURONIC               0.200% Clear                           Slt. Haze                                -- --  1.04                                          Marginal       F68                                Stability    27 PLURONJC               0.300% Clear                           Slt. Haze                                -- --  1.10                                          Marginal       F68                                Stability    28 PLURONIC               0.500% Clear                           Slt. Maze                                -- --  1.08                                          Marginal       F68                                Stability    29 PLURONIC               0.070% Clear                           Clear                                1.06                                   1.08                                       0.97                                          Marginal       P123                               Stability    30 BRIJ 30/               0.05%/ Clear                           Clear                                1.04                                   1.01                                       1.01                                          Stable       PS-80   0.063%    31 PLUR F68/               0.1%/  Clear                           Clear                                1.05                                   1.06                                       1.10                                          Stable       PS-80   0.1%    32 PLUR F68/               0.1%/  Clear                           Clear                                1.05                                   1.05                                       1.03                                          Stable       BRIJ 30 0.03%    33 PLUR F68/               0.1%/  Clear                           Clear                                1.06                                   1.04                                       1.05                                          Stable       BRIJ 30 0.01%    34 PLURONIC               0.100% Cloudy                           Cloudy                                1.07                                   0.87                                       0.56                                          Unstable       F88    35 PLURONIC               0.100% Cloudy                           Cloudy                                1.04                                   0.77                                       0.39                                          Unstable       F98    36 PLURONIC               0.100% Clear                           Cloudy                                1.04                                   0.87                                       0.55                                          Unstable       F108    37 PLURONIC               0.100% Clear                           Clear                                1.06                                   1.04                                       0.98                                          Marginal       F127                               Stability    38 PLUR F68/               0.075%/                      Clear                           Clear                                1.12                                   --  1.11                                          Stable       BRIJ 30 0.01%    39 PLUR F68/               0.05%/ Clear                           Clear                                1.12                                   --  1.09                                          Stable       BRIJ 30 0.01%    40 PLUR F68/               0.025%/                      Clear                           Clear                                1.10                                   --  1.04                                          Stable       BRIJ 30 0.01%    41 PLUR F68/               0.01%/ Cloudy                           Cloudy                                1.07                                   --  0.64                                          Unstable       BRIJ 30 0.01%    42 PLURONIC               0.100% Clear                           Clear                                1.12                                   --  0.93                                          Marginal       F127                               Stability    43 PLURONIC               0.075% Clear                           Clear                                1.10                                   --  0.61                                          Unstable       F127    44 PLURONIC               0.050% Clear                           Slt. Haze                                1.09                                   --  0.20                                          Unstable       F127    45 PLURONIC               0.025% Slt. Haze                           Cloudy                                1.07                                   --  0.00                                          Unstable       F127    46 PLURONIC               0.010% Cloudy                           Cloudy                                1.06                                   --  0.00                                          Unstable       F127    47 PLUR F68/               0.05%/ Clear                           Clear                                1.04                                   --  1.01                                          Stable       BRIJ 30 0.01%    48 PLUR F68/               0.05%/ Clear                           Clear                                1.01                                   --  1.01                                          Stable       BRIJ 30 0.008%    49 PLUR F68/               0.05%/ Clear                           Clear                                1.00                                   --  1.03                                          Stable       BRIJ 30 0.005%    50 PLUR F68/               0.03%/ Clear                           Clear                                1.06                                   --  0.99                                          Marginal       BRIJ 30 0.008% Stability    51 PLUR F68/               0.03%/ Clear                           Cloudy                                1.01                                   --  0.79                                          Unstable       BRIJ 30 0.005%    52 PLUR F68/               0.1%/  Clear                           Clear                                1.14                                   --  1.11                                          Stable.       PS-80   0.05%                      A few specks.    53 PLUR F68/               0.1%/  Clear                           Clear                                1.14                                   --  1.11                                          Stable.       PS-80   0.01%                      A few specks.    54 PLUR F68/               0.1%/  Clear                           Clear                                1.15                                   --  1.10                                          Stable.       PS-80   0.005%                     A few specks.    55 PLUR F68/               0.1%/  Clear                           Clear                                1.14                                   --  1.09                                          Stable.       PS-80   0.001%                     A few specks.    56 PLUR F68/               0.1%/  Clear                           Cloudy                                1.12                                   1.09                                       1.02                                          Unstable       PS-80   0.0005%    57 PLUR F68/               0.1%/  Slt. Haze                           Cloudy                                1.09                                   1.09                                       1.02                                          Unstable       PS-80   0.0001%    58 PLUR F68/               0.05%/ Clear                           Cloudy                                1.08                                   1.00                                       0.72                                          Unstable       PS-80   0.001%    __________________________________________________________________________

EXAMPLE 3

In this example, a study was conducted to compare the efficacy of rBPI₂₃formulated with and without the preferred formulation of the inventionin an actinomycin-D sensitized mouse model according to Pieroni et al.,Proc. Soc. Exp. Biol. & Med.; 133, 790 (1970). According to thisexample, ICR mice were administered an intravenous injection ofactinomycin-D (800 μg/kg). Immediately thereafter, groups of 15 miceeach received an injection of one of several doses of rBPI₂₃ BR-1!characterized by about 1 to about 199 of the first 199 amino acids ofthe mature human BPI molecule and produced according to the methods ofTheofan et al., U.S. patent application Ser. No. 08/013,801 filed Feb.2, 1993 at 1 mg/mL in citrate buffered saline (0.2M citrate, 0.15M NaCl,pH 5.0). The mouse injections were at dosages of 0.03, 0.1, 1.0 and 3.0mg/kg. As a control, some animals received the formulation buffer withor without the poloxamer and polysorbate surfactants. Deaths wererecorded over seven days.

The results are shown in FIGS. 1 and 2. FIG. 1 shows the number of micesurviving on each study day in the buffer and 3.0 mg/kg rBPI₂₃ treatmentgroups. For both buffer groups (with or without poloxamer andpolysorbate surfactants), mortality was 80% overall. In contrast, rBPI₂₃in the presence of excipients was even more potent than either buffer orrBPI₂₃ without excipients. FIG. 2 summarizes the data for the differentdose groups at day 7 (final survivors). Beginning at the 0.1 mg/kg doselevel, rBPI₂₃ formulated with the preferred surfactant formulationsprovided significantly greater protection to the lethal effects of LPS(P<0.05 or better) than did rBPI₂₃ in the absence of added excipients.

EXAMPLE 4

In this example, experiments were conducted to determine the turbidityof various rBPI-containing pharmaceutical compositions with and withoutthe preferred surfactant formulation of the invention. In this context,turbidity refers to the tendency of pharmaceutical compositions toengage in unfolding (i.e. loss of tertiary protein structure) and/orparticle formation (interactions between individual proteins to formlarger (>10 μm) particles). The pharmaceutical compositions testedcontained either rBPI(1-199)ala¹³², rBPI(1-193)ala¹³² or various samplesof rBPI₂₃ produced according to co-owned and co-pending U.S. patentapplication Ser. No. 08/013,801 filed Feb. 2, 1993 in either a citratebuffer (20 mM sodium citrate, 150 mM sodium chloride, pH 5.0) or acitrate buffer containing 0.1% poloxamer 188 and 0.002% polysorbate 80.

Samples were analyzed to determine their resistance to turbidity overtime at increasing temperature and at pH 7.0. Prior to analysis, allsamples were diluted to a concentration of 0.1 mg/mL in 50 mM potassiumphosphate at pH 7.0. Turbidity measurements were obtained by placingsamples in quartz cuvettes for use in a Shimadzu UV-160 UV-V isspectrophotometer equipped with a temperature-controlled cuvette holderattached to a recirculating water bath. Upon equilibrating the cuvetteholder at 57° C., absorbance at 280 nm was measured to confirm thatsamples had been diluted to the proper concentration. Following this,the absorbance of samples at 350 nm was measured every 2 minutes for 1hour to determine the change in absorbance over time.

Results are presented in FIG. 3 showing a lower rate of change inturbidity (i.e., a lower rate of increase in absorbance over time),indicating increased stability against the formation of particles. Asshown in FIG. 3, the addition of the preferred combination ofsurfactants resulted in increased stability (resistance to particleformation) of all compositions tested. Moreover, the rBPI(1-199)ala³²and rBPI(1-193)ala¹³² exhibited greatly improved resistance to particleformation relative to wild-type compositions rBPI₂₃ !.

EXAMPLE 5

In this example surface tension measurements were made of polysorbateand poloxamer surfactants or combinations of the two in solutions of theBPI protein product rBPI₂₁ Δcys according to the procedure set out inthe Kruss Digital Tensiometer K10ST Users Manual, Chapter 4: Measuringwith the Plate. A decrease in surface tension indicates an increase inthe surface activity of the surfactant, which has conventionally beenthought to be the mechanism by which surfactants stabilize proteins.These procedures established that poloxamer surfactants provideadvantageous results by a different and unexpected mechanism.

Specifically, a 2 mg/mL solution of unformulated rBPI₂₁ Δcys (lot 30216)was diluted with 20 mM sodium citrate, 150 mM sodium chloride, pH 5.0rendering a 1 mg/mL solution. 15 mL of this solution was placed into a50 mL glass beaker containing a mini stir bar. Surfactants poloxamer188, polysorbate 80, or combinations of both were added incrementally upto 0.10%. Before each surface tension measurement, the platinum platewas heated above the reducing zone (blue flame) of a gas burner untilthe plate just began to glow red. The platinum plate was heated forabout 10 to 15 seconds while turning the plate from side to side andthen suspended back into the instrument. Each addition of surfactant wasgently mixed using a magnetic stirrer and the solution was allowed tostand for 2 minutes on the thermostat vessel equilibrated at 4.6° C. Thevalue for the surface tension was read after five minutes.

The first part of this experiment evaluated the surface activity of thesurfactants alone in buffer. Using the citrate saline buffer (20 mMsodium citrate, 150 mM sodium chloride, pH 5.0) as the baseline,surfactants were added incrementally. FIG. 4 is a plot of surfacetension dependence on surfactant concentrations; the corresponding datais presented in Table 3. The open squares represent the citrate salinebuffer in varying concentrations of poloxamer 188 while the closedcircles represent the same buffer in varying concentrations ofpolysorbate 80. The citrate-saline buffer solution alone had a surfacetension of about 75 mN/m at 4.6° C., similar to H₂ O. With increasingconcentrations of surfactants, the buffer solution showed decreasingsurface tension. With 0.10% poloxamer 188, the surface tension of thesolution was 55 mN/m. On the other hand, with 0.10% polysorbate 80, thesurface tension of the solution was 45 mN/m. The decrease in surfacetension indicates an increase in the surface activity of the surfactant,i.e., the lower the surface tension, the higher the surface activity.The results indicate that polysorbate 80 is more surface active thanpoloxamer 188.

In the second part of the experiment, the surface activity of rBPI₂₁Δcys in the presence of surfactants was evaluated. The results show thatrBPI₂₁ Δcys at 1 mg/mL in citrate saline buffer, pH 5.0, is surfaceactive with a surface tension of about 54 mN/m at 4.6° C. The additionof polysorbate 80 (PS80) alone up to 0.0005% did not change the surfacetension of rBPI₂₁ Δcys solution either (FIG. 4, closed triangles). Atconcentrations of polysorbate 80 exceeding 0.0005%, the surface tensionof rBPI₂₁ Δcys follows that of buffer with PS80 alone (no BPI), in whichthe surface tension of the solution decreases as the concentration ofpolysorbate 80 is gradually increased. For buffer with PS80 alone, thesurface tension of 54 mN/m was reached when the PS80 concentration wasincreased from 0.0005%. These results indicate that when PS80concentration is less than 0.0005%, the surface activity of the solutionis dominated by rBPI₂₁ Δcys. On the other hand, at PS80 concentrationabove 0.0005%, the surface activity of the solution is modulated bypolysorbate 80 the addition of poloxamer 188 (F68) alone to rBPI₂₁ Δcysup to 0.10% did not change the surface activity of rBPI₂₁ Δcys solutionsignificantly (FIG. 4, open triangles).

                                      TABLE 3    __________________________________________________________________________                                    8          2        4        6       ΔCys +                                              10          Buffer + Buffer + ΔCys +                                    0.1% F68 +                                              ΔCys +    1     F68  3   PS80 5   F68 7   PS80  9   PS80    % F68 (mN/m)               % PS80                   (mN/m)                        % F68                            (mN/m)                                % PS80                                    (mN/m)                                          % PS80                                              (mN/m)    __________________________________________________________________________    1 0.00000          75.4 0.00000                   75.1 0.00000                            54.2                                0.00000                                    53.7  0.00000                                              54.9    2 0.00001          74.9 0.00001                   66.8 0.00001                            54.7                                0.00001                                    53.4  0.00001                                              55.0    3 0.00003          74.3 0.00002                   60.0 0.00002                            54.2                                0.00002                                    53.3  0.00002                                              53.2    4 0.00005          68.2 0.00003                   60.0 0.00003                            54.9                                0.00003                                    53.9  0.00003                                              53.3    5 0.00007          65.9 0.00005                   60.0 0.00004                            54.8                                0.00004                                    53.9  0.00004                                              52.8    6 0.00010          64.0 0.00007                   57.4 0.00005                            55.0                                0.00005                                    53.5  0.00005                                              52.4    7 0.00013          65.8 0.00010                   56.6 0.00006                            55.2                                0.00006                                    53.5  0.00006                                              53.3    8 0.00015          65.4 0.00015                   57.2 0.00007                            55.4                                0.00007                                    53.4  0.00007                                              53.6    9 0.00017          66.5 0.00020                   56.7 0.00008                            54.8                                0.00008                                    53.8  0.00008                                              53.8    10      0.00020          65.7 0.00050                   55.6 0.00009                            55.0                                0.00010                                    53.4  0.00009                                              53.2    11      0.00023          66.0 0.00070                   55.3 0.00010                            54.9                                0.00020                                    53.5  0.00010                                              53.5    12      0.00027          64.4 0.00100                   54.2 0.00030                            55.3                                0.00030                                    53.2  0.00020                                              53.2    13      0.00030          63.8 0.00300                   52.7 0.00050                            54.5                                0.00050                                    52.3  0.00030                                              53.0    14      0.00033          64.1 0.00700                   49.2 0.00070                            55.5                                0.00070                                    51.5  0.00050                                              52.0    15      0.00037          63.1 0.01000                   48.3 0.00100                            54.9                                0.00100                                    51.0  0.00070                                              51.2    16      0.00040          64.2 0.03000                   46.5 0.00500                            54.9                                0.00200                                    50.6  0.00100                                              50.5    17      0.00043          61.8 0.07000                   45.3 0.01000                            55.4                                0.00500                                    50.1  0.00130                                              50.4    18      0.00047          62.4 0.10000                   45.4 0.05000                            53.6                                0.01000                                    48.6  0.00170                                              49.8    19      0.00050          63.5          0.10000                            53.7                                0.05000                                    45.6  0.00200                                              48.8    20      0.00060          61.6                  0.10000                                    45.0  0.00500                                              47.7    21      0.00070          62.5                            0.01000                                              46.7    22      0.00080          62.0                            0.05000                                              45.4    23      0.00100          61.7                            0.10000                                              45.0    24      0.00300          61.2    25      0.00500          59.3    26      0.00700          58.9    27      0.01000          58.4    28      0.03000          56.6    29      0.07000          56.1    30      0.10000          55.1    __________________________________________________________________________

EXAMPLE 6

Protein samples were analyzed by Differential Scanning Calorimetry (DSC)to study the unfolding (or denaturation) of the protein. The startingmaterials for DSC analysis were identical to those used in the surfacetension measurement. A series of rBPI₂₁ Δcys solutions was prepared withvarying concentrations of surfactants, poloxamer 188, polysorbate 80 orcombinations of both, and diluted with buffer (20 mM sodium citrate, 150mM sodium chloride, pH5.0) to give a final rBPI₂₁ Δcys concentration of1 mg/mL. A series of buffer solutions was also prepared with surfactantsat the same concentrations as in the rBPI₂₁ Δcys solutions to serve asblanks for DSC. Each solution was filtered and placed into a 2 mLsterile plastic vial. The samples were packed into a 4° C. cold boxuntil subjected to DSC Analysis.

The behavior of rBPI₂₁ Δcys was evaluated as the temperature of thesolution was gradually increased from ambient temperature to about 90°C., at a rate of 1° C. per minute. As the temperature is increased twoevents occur. The first event is an unfolding reaction, which isendothermic, and is illustrated by an upward peak in the scans. Thesecond event is precipitation, which is exothermic, and is depicted by adownward peak in the scans. In the scans depicted in FIGS. 5, 6 and8-10, each scan is offset to facilitate analysis of data. In the rBPI₂₁Δcys solution not containing surfactants (FIG. 5, Scan 1) the unfoldingof the protein at 65° C. was followed immediately by the second event,precipitation of the protein at 66 to 67° C.

With low poloxamer 188 (PLURONICO F68) concentrations ranging between0.001% to 0.01%, the unfolding and precipitation events are similar tothe rBPI₂₁ Δcys solution without surfactants (FIG. 5, Scans 2 to 5),i.e. as rBPI₂₁ Δcys unfolds, precipitation takes place immediately. Withpoloxamer 188 concentrations exceeding 0.05%, the unfolding of rBPI₂₁Δcys still occurs at 65° C., but precipitation does not occur until thetemperature reaches 85° C. (FIG. 5, Scan 6). FIG. 6 shows that atpoloxamer 188 concentrations between 0.01% and 0.05%, there is a gradualtransition of delayed precipitation of unfolded BPI. These resultssuggest that at poloxamer 188 concentrations higher than 0.01%, unfoldedrBPI₂₁ Δcys can be stabilized and the occurrence of precipitation isdelayed. A plot of denaturation and precipitation temperature dependenceover the surfactant (poloxamer 188) concentration is shown in FIG. 7.The effects of poloxamer 188 appear to delay the precipitation of rBPI₂₁Δcys to a higher temperature but not to stabilize its native structureas the T_(m) (denaturation temperature) and ΔH (energy of denaturation)did not change.

rBPI₂₁ Δcys formulated with polysorbate 80 at concentrations up to 1%was likewise analyzed. The isotherms were similar to rBPI₂₁ Δcyssolution without surfactants (FIG. 8: Scans 1 and 8-13, FIG. 9: Scans11, 12). Polysorbate 80 did not maintain the rBPI₂₁ Δcys in solution athigher temperatures. The stabilization of unfolded rBPI₂₁ Δcys is thusunique to poloxamer 188.

The two formulations using combined poloxamer 188 and polysorbate 80,namely 0.1%F68/0.001%PS80 and 0.1%F68/0.002%PS80, showed the same scanprofile as rBPI₂₁ Δcys containing 0.05% and 0.1% PLURONIC F68, withunfolding at 65° C. and precipitation at 85° C. (FIG. 8: Scans 14, 15).

In addition to determining the melting behavior of rBPI₂₁ Δcys,rescanning was done with rBPI₂₁ Δcys formulations containing 0.05% and0.10% poloxamer 188 to determine if unfolding is a reversible process.The temperature of the rBPI₂₁ Δcys solution was first increased to 75°C. (temperature after denaturation/unfolding but before precipitation),then was cooled down for repeat scanning. FIG. 10 shows that theaddition of poloxamer 188 to rBP₂₁ Δcys does not make unfoldingreversible. Profiles A5,1 and A6,1 show the scanning to 75° C., whileprofile A5,2 and A6,2 are repeat scanning after cooling the system from75° C. if unfolding were a reversible process, 6 and 7 scan profileswould have been obtained.

The experimental results described above demonstrate that poloxamersurfactant alone is capable of stabilizing BPI-related polypeptides insolution and delaying the occurrence of precipitation by a mechanismthat does not appear to involve modulation of the surface tension of theaqueous solution. This property is unique to poloxamer because othersurfactants such as polysorbate 80 do not affect the precipitationphenomenon and do involve modulation of the surface tension of theaqueous solution.

EXAMPLE 7

The rate of rBPI₂₁ Δcys precipitation during shipping was simulated inthe laboratory by adjustment of the speed of the horizontal shaker.During five cycles of surface shipping, about 70% of the unformulated(surfactant free) rBPI₂₁ Δcys precipitated. By varying the speed (rpm)of the flat-bed shaker, shake tests were then constituted such that 70to 90% of unformulated rBPI₂₁ Δcys subjected to the shake testprecipitated. No rBPI₂₁ Δcys was precipitated when the unformulatedproduct was shaken on a flat bed shaker at 110 rpm or less for 18 hoursat 4° C. Shaking at 140 rpm (rather than at 150 rpm as in Examples 1 and2) most closely simulated the agitation occurring during five cycles ofsurface transport. Changes in the flow dynamics of the liquid in thevial are substantially different at 140 rpm versus 150 rpm. Compositionsincluding various concentrations of surfactant combinations werescreened using the 140 rpm shake condition and the results obtained areset out in Table 4. It was determined that the optimal surfactantconcentrations for protection from precipitation were 0.2% poloxamer 188with 0.002% polysorbate 80 and 0.15% poloxamer 188 with 0.005%polysorbate 80.

                  TABLE 4    ______________________________________    Summary of Shake Test at 140 rpm for rBPI.sub.21 Δcys at 4°    C.                              Concentration    Poloxamer            PS80    Visual    by MA7C HPLC (mg/ml)    188(%)  (%)     (see note)                              Before After Loss (%)    ______________________________________            0.005   5         2.14   1.54  28    0.075   0.010   4         2.10   1.56  26            0.020   1         2.14   1.68  21            0.002   5         2.24   1.85  17            0.005   4         2.14   1.85  14    0.100   0.010   1         2.10   1.87  11            0.020   1         2.13   1.94  9            0.002   3         2.19   1.92  12            0.005   2         2.08   1.95  6    0.150   0.010   1         2.19   1.94  11            0.020   1         2.06   1.96  5            0.002   2         2.19   1.98  10    0.200   0.005   1         2.19   1.95  11            0.005   5         2.14   1.54  28            0.010   1         2.22   1.95  12    ______________________________________     Note: The scoring for visual observation is as follows:     1. Clear     2. Clear with few particulates     3. Slightly hazy     4. Hazy     5. Cloudy

Based on the above data, a preferred formulation for 2 mg/mL rBPI₂₁ Δcysto be stored at 4° C. would contain 5 mM citrate, 150 mM NaCl, pH 5.0,0.2% poloxamer 188 and 0.002% polysorbate 80. An alternative formulationfor 2 mg/ml rBPI₂₁ Δcys to be stored at 4° C. would contain 5 mMcitrate, 150 mM NaCl, pH 5.0, 0.15% poloxamer 188 and 0.005% polysorbate80.

In summary, aggregation precipitation is one of the major causes ofprotein instability and can occur when proteins at the air-Liquidinterface unfold and expose hydrophobic domains. If left unprotected,proteins self-associate through the interaction of the exposedhydrophobic domains, resulting in aggregation and/or precipitation. Withthe use of the surfactants and surfactant combinations of the invention,protein can be stabilized in two ways. First, exposed hydrophobicregions at the air-liquid interface are shielded by poloxamersurfactants. Second, additional stabilization can be provided bypolysorbate surfactants through conventional modulation of the surfaceactivity of the solution.

Numerous modifications and variations of the above-described inventionare expected to occur to those of skill in the art. Accordingly, onlysuch limitations as appear in the appended claims should be placedthereon.

What is claimed is:
 1. A pharmaceutical composition comprising a BPIprotein or BPI fragment thereof, or a BPI analog of said BPI protein orBPI fragment, in combination with a poloxamer surfactant in asolubilizing or stabilizing concentration, wherein said poloxamersurfactant is poloxamer 188 and is present at a solubilizing orstabilizing concentration of from about 0.01% to about 1% by weight. 2.The pharmaceutical composition of claim 1 wherein the poloxamersurfactant is present at a solubilizing or stabilizing concentration offrom about 0.0% to about 0.5% by weight.
 3. A pharmaceutical compositioncomprising a bactericidal/permeability-increasing (BPI) protein or BPIfragment thereof, or a BPI analog of said BPI protein or BPI fragment,in combination with a polyoxypropylene-polyoxyethylene block copolymer(poloxamer) surfactant in a solubilizing or stabilizing concentration,wherein said poloxamer surfactant is poloxamer 403.